Assays, antibodies, and standards for detection of oxidized and MDA-modified low density lipoproteins

ABSTRACT

Immunoassays for malondialdehyde-modified low density lipoprotein (MDA-modified LDL) and oxidized low density lipoprotein (OxLDL), monoclonal antibodies (and the cell lines for them) for use in the assays, and a storage-stable standard (which may be used as a calibrator and/or control) are disclosed. MDA-modified LDL and OxLDL are implicated in atherosclerosis and its etiology.

BACKGROUND

[0001] The present invention relates to assays, antibodies (particularlymonoclonal antibodies), and standards for detection (i.e., determinationof the presence and/or quantitation of the amount) of oxidized lowdensity lipoprotein (OxLDL) and malondialdehyde-modified low densitylipoprotein (MDA-modified LDL) in samples, the samples typically beingderived from body fluids or tissues.

[0002] Lipoproteins are multicomponent complexes of protein and lipids.Each type of lipoprotein has a characteristic molecular weight, size,chemical composition, density, and physical role. The protein and lipidare held together by noncovalent forces.

[0003] Lipoproteins can be classified on the basis of their density asdetermined by ultracentrifugation. Thus, four classes of lipoproteinscan be distinguished: High Density Lipoproteins (HDL), IntermediateDensity Lipoproteins (IDL), Low Density Lipoproteins (LDL), and Very LowDensity Lipoproteins (VLDL).

[0004] The purified protein components of a lipoprotein particle arecalled apolipoproteins (apo). Each type of lipoprotein has acharacteristic apolipoprotein composition. In LDL the prominentapolipoprotein protein is apo B-100. Apo B-100 is one of the longestsingle chain polypeptides known and consists of 4536 amino acids. Ofthese amino acids the lysine residues or moieties (there are 356 suchlysine residues or moieties) can be substituted or modified by aldehydes(e.g., malondialdehyde).

[0005] Oxidation of the lipids in LDL (whether in vitro, e.g., bycopper-induced oxidation, or whether in vivo) results in the generationof reactive aldehydes, which can then interact with the lysine residuesor moieties of apo B-100. The outcome of this lysine substitution ormodification is that the resulting OxLDL, which is also MDA-modifiedLDL, is no longer recognized by the LDL receptor at the surface offibroblasts but by scavenger receptors at the surface of macrophages. Atleast 60 out of the 356 lysines (or lysine residues or moieties) of apoB-100 have to be substituted in order to be recognized by the scavengerreceptors (see document number 1 of the documents listed near the end ofthis application, all of which documents are hereby incorporated intheir entireties for all purposes). The uptake of such OxLDL bymacrophages results in foam cell generation, which is considered to bean initial step in atherosclerosis.

[0006] Endothelial cells under oxidative stress (e.g., in acutemyocardial infarction patients) and activated blood platelets alsoproduce aldehydes, which interact with the lysine moieties in apo B-100,resulting in the generation of aldehyde-modified LDL that is alsorecognized by the scavenger receptors. However, the lipids in thisaldehyde-modified LDL are not oxidized. Enzymatic activity inmacrophages (e.g. myeloperoxidase) results in the oxidation of both thelipid and the protein moieties of LDL. All these pathways result inaldehyde-type modification of the protein moiety of LDL.

[0007] In vitro experiments and experiments in animal models havesuggested that OxLDL and/or aldehyde-modified LDL may contribute to theprogression of atherosclerosis by inducing endothelial dysfunction, foamcell generation, smooth muscle cell proliferation, and plateletactivation (for review see document number 2). A positive correlationbetween the levels of autoimmune antibodies that cross-react withaldehyde-modified LDL and the progression of carotid atheroscleroticlesions in patients suggested that OxLDL and/or aldehyde-modified LDLmight contribute to the progression of human atherosclerosis (seedocument 3).

[0008] However, the possibility that the autoimmune antibodies weredirected against other aldehyde-modified proteins, e.g., albumin, couldnot be excluded. Therefore, the contribution of OxLDL andaldehyde-modified LDL (whether or not resulting from oxidation of thelipid moiety) to human atherosclerosis may be able to be establishedwhen non-invasive tests that are specific for these substances (i.e.,have high affinity for those substances in preference to othersubstances) become available.

[0009] Because the underlying mechanisms of oxidation of LDL may bedifferent in different patient populations (e.g., in diabetes patients,chronic renal failure patients, heart transplant patients) and becauseat least some of the mechanisms may be independent of lipid oxidation,such tests should be specific for both OxLDL and aldehyde-modified LDL(e.g., MDA-modified LDL) and thus preferentially be based on thedetection of conformational changes that specifically occur in the apoB-100 moiety of LDL following aldehyde-type substitution of lysineresidues. In other words, there is a need for such non-invasive tests(i.e., assays) that are highly specific for the analytes of interest(i.e., MDA-modified LDL and OxLDL). There is also a need for antibodiesthat are specific for the analytes of interest. There is also a need fora stable standard (e.g., to be used as calibrator and/or control) forthe assays.

SUMMARY OF THE INVENTION

[0010] An invention satisfying those needs and having other features andadvantages that will be apparent to those skilled in the art has nowbeen developed. The present invention provides antibody-based assaysthat are capable of specifically quantitating (quantifying) both OxLDLand aldehyde-modified LDL or MDA-modified LDL in samples, e.g., samplesderived from body fluids (like plasma or serum) or tissues. The presentinvention also provides monoclonal antibodies useful in those assays andcell lines (hybridomas) that produce those antibodies. The presentinvention also provides a storage-stable standard, which can be used asa calibrator and as a control for the assays. Having such a standard isnecessary for having reliable and reproducible and therefore usefulassays.

[0011] Broadly, in one aspect the present invention concerns animmunological assay for the detection and/or quantification ofMDA-modified LDL and OxLDL in a sample, said assay comprising:

[0012] a) contacting the sample with a first antibody that has highaffinity for MDA-modified LDL and OxLDL; and

[0013] b) thereafter visualizing and/or quantifying a binding reactionbetween the first antibody and the MDA-modified LDL and OxLDL present inthe sample;

[0014] wherein the MDA-modified LDL and OxLDL for which the firstantibody has high affinity contain at least 60 substituted lysinemoieties per apo B-100 moiety.

[0015] That assay may, for example, be a competitive assay, a sandwichassay, an immunohistochemical assay, etc. “Competitive assays” arewell-known and any competitive assay may be used in this inventionprovided it is within the limitations of the invention and that thebenefits of the invention can be achieved. “Sandwich assays” arewell-known and any sandwich assay may be used in this invention providedit is within the limitations of the invention and that the benefits ofthe invention can be achieved. “Immunohistochemical assays” arewell-known and any immunohistochemical assay may be used in thisinvention provided it is within the limitations of the invention andthat the benefits of the invention can be achieved.

[0016] In another aspect, the present invention concerns animmunological sandwich assay for the detection and/or quantification ofMDA-modified LDL in a sample in which assay a first antibody that has ahigh affinity for MDA-modified LDL is bound to a substrate, said assaycomprising:

[0017] (a) contacting the sample with the substrate having bound to itthe first antibody under binding conditions so that at least some of anyMDA-modified LDL in the sample will bind to the first antibody;

[0018] (b) thereafter removing unbound sample from the substrate;

[0019] (c) thereafter contacting the substrate with a second antibodythat has a high affinity for MDA-modified LDL; and

[0020] (d) thereafter visualizing and/or quantifying the MDA-modifiedLDL that was present in the sample;

[0021] wherein the MDA-modified LDL for which the first antibody and thesecond antibody have high affinity contains at least 60 substitutedlysine moieties per apo B-100 moiety.

[0022] As used herein (including the claims), “high affinity” means anaffinity constant (association constant) of at least about 5×10⁸ M⁻¹,desirably at least about 1×10⁹ M⁻¹, preferably at least about 1×10¹⁰M⁻¹,and most preferably of at least about 1×10¹¹M⁻¹. As used herein(including the claims), “low affinity” means an affinity constant(association constant) of less than about 1×10⁷ M⁻¹, desirably less thanabout 1×10⁶M⁻¹, and preferably less than about 1×10⁵M⁻¹. Affinityconstants are determined in accordance with the appropriate methoddescribed in Holvoet et al. (4).

[0023] The antibodies that can be used in this invention will bind withMDA-modified LDL and/or OxLDL whose apo B-100 moieties contain at least60, desirably at least about 90, more desirably at least about 120,preferably at least about 180, more preferably at least about 210, andmost preferably at least about 240 substituted lysine residues per apoB-100 moiety. The range of lysine substitution will generally be from 60to about 240 and preferably from about 120 to about 240 substitutedlysine moieties per apo B-100 moiety.

[0024] Each new monoclonal antibody is highly specific for aconformational epitope that is present when at least about 60,preferably at least about 120 lysine residues, are substituted and byvirtue thereof can distinguish various markers or indications related toatherosclerosis. Antibodies recognizing epitopes present when less thanabout 60 lysines are substituted or modified are less specific but arestill useful (e.g., they may be used as the secondary antibody in asandwich ELISA).

[0025] The preferred antibodies used herein are monoclonal antibodiesmAb-4E6, mAb-1h11, and mAb-8A2. Their affinity constants for native LDL,MDA-modified LDL, and OxLDL are as follows: Antibody Native LDLMDA-modified LDL OxLDL mAb-4E6 less than 3 × 10¹⁰ 2 × 10¹⁰ 1 × 10⁶mAb-1H11 less than 3 × 10¹⁰ less than 1 × 10⁶ 1 × 10⁶ mAb-8A2 5 × 10⁹ 1× 10¹⁰ 1 × 10¹⁰

[0026] In yet another aspect, the present invention concerns (a)monoclonal antibody mAb-4E6 produced by hybridoma Hyb4E6 deposited atthe BCCM under deposit accession number LMBP 1660 CB on or about Apr.24, 1997, (b) monoclonal antibody mAb-8A2 produced by hybridoma Hyb8A2deposited at the BCCM under deposit accession number LMBP 1661 CB on orabout Apr. 24, 1997, (c) hybridoma Hyb4E6 deposited at the BCCM underdeposit accession number LMBP 1660 CB on or about Apr. 24, 1997, and (d)hybridoma Hyb8A2 deposited at the BCCM under deposit accession numberLMBP 1661 CB on or about Apr. 24, 1997.

[0027] The antibodies used in the assays of this invention arepreferably those two (i.e., mAb-4E6 and mAb-8A2) as well as mAb-1H11.The cell line for antibody mAb-1H11 is produced by hybridoma Hyb1H11,which was deposited at the BCCM under deposit accession number LMBP 1659CB on or about Apr. 24, 1997.

[0028] The BCCM is the Belgian Coordinated Collections of Microorganismsauthorized by the “Budapest Treaty of 28 Apr. 1977 on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure.” Its address is c/o The University of Gent, K.Ledeganckstraat 35, B-9000 Gent, Belgium.

[0029] The assay may be of a type that is well-known, such as anEnzyme-Linked Immunosorbent Assay (ELISA). For example, in the case of asandwich ELISA, mAb-4E6 (for MDA-modified LDL and OxLDL) or mAb-1H11(for MDA-modified LDL) may be bound to a solid substrate andsubsequently contacted with a sample to be assayed. After removal of thesample, binding between the specific antibody and OxLDL and/orMDA-modified LDL captured out of the sample can be visualized and/orquantified by detection means. Detection means may be a labeled, lessspecific secondary antibody that recognizes a different part of the apoB-100 moiety of the captured analyte (e.g., mAb-8A2).

[0030] In the case of a competitive ELISA, a solid substrate coated withOxLDL or MDA-modified LDL may be contacted for a predetermined period oftime with the monoclonal antibody mAb-4E6 and a sample thought or knownto contain OxLDL and/or MDA-modified LDL, after which period of timeunbound antibody and sample are removed and a binding reaction betweenantibody and OxLDL and/or MDA-modified LDL bound to the substrate isvisualized and/or quantified. Quantification in a competitive ELISA isindirect because the binding between the antibody and the analyte in thesample is not measured but instead the amount of antibody that binds tothe known amount of OxLDL or MDA-modified LDL that is coated on (boundto) the substrate is measured. The more antibody bound to the knownamount of OxLDL or MDA-modified LDL coated on the substrate, the lessanalyte there was in the sample.

[0031] In yet another aspect, the present invention concerns a stablestandard containing MDA-modified LDL whose extent of substitution of itslysine moieties will remain essentially constant, over normal periods oftime during normal storage for biological materials, the MDA-modifiedLDL of said standard being made by contacting (incubating)malondialdehyde with LDL at a predetermined molar ratio ofmalondialdehyde to the apo B-100 moiety of the LDL.

[0032] “Over normal periods of time during normal storage for biologicalmaterials” as used herein refers to the time periods and conditionsunder which biological materials to be used in assays and otherlaboratory work are typically stored. Those conditions will typicallyinclude low temperature and in appropriate cases freezing, either withor without lyophilization. Depending on the particular biologicalmaterial, if the material is stored under the appropriate temperatureand other conditions (e.g., lack of vibration or other movement, properhumidity), the material may be stable for at least three months,desirably for over a year, preferably for over two years, and mostpreferably for over three years.

[0033] The standard preferably contains an agent that reduces theability of any metal ions present to catalyze oxidation of the LDL(e.g., a chelating agent, such as EDTA) and/or one or more anti-oxidants(e.g., BHT and/or Vitamin E). Preferably both the agent that reduces theability of any metal ions present to catalyze oxidation of the LDL andthe anti-oxidant are used. It has surprisingly been found that whenusing an antibody that is specific for both OxLDL and MDA-modified LDL,the storage-stable standard of this invention (containing MDA-modifiedLDL and not OxLDL) can be used. That eliminates the need to try toformulate, store, and use a stable standard containing OxLDL. OxLDL maycontinue to oxidize under typical storage conditions, making using as astandard a composition containing OxLDL difficult if not almostimpossible. EDTA will typically be used in concentrations of 0.5 to 5mM, preferably in concentrations of 0.5 to 2 mM. BHT will typically beused in concentrations of 5 to 50 μM, preferably in concentrations of 10to 20 μM. Vitamin E will typically be used in concentrations of 5 to 50μM, preferably in concentrations of 10 to 20 μM. The standard may alsocontain anti-platelet agents and coagulation inhibitors.

[0034] It has been found that LDL that has been modified by treatmentwith MDA is highly stable. Such MDA-modified LDL (which is not oxidized,i.e., its lipid moiety is not oxidized) could be added to referenceplasma samples and those samples could be frozen and thawed withoutincreasing the extent of lysine substitution. Because the total numberof lysine residues in all apo B-100 molecules is identical, a constantMDA/apo B-100 molar ratio in the reaction mixture will result in anidentical number of substituted lysines in the MDA-modified LDL. Incontrast, for example, metal-ion mediated oxidation of LDL ultimatelyresults in a variable extent of lysine substitution because it dependson the oxidation sensitivity of the LDL preparation, which by itselfdepends on fatty acid composition and antioxidant content, which arehighly variable even in healthy control individuals.

[0035] As described below, a correlation between the oxidation of LDLand the extent of post-transplant atherosclerosis in heart transplantpatients was established using this invention. The relationship betweenendothelial injury and the modification of LDL was established inchronic renal failure patients that are at high risk for atheroscleroticcardiovascular disease. It was also demonstrated that endothelial injuryis an initial step in atherosclerosis.

[0036] Based on the characteristics of the oxidatively modified LDL fromthe plasma of heart transplant and chronic renal failure patients, itwas concluded that cell-mediated aldehyde modification independent oflipid oxidation was at least partially involved. This finding furthersupported the hypothesis that an assay for oxidatively modified LDL hasto detect both OxLDL and aldehyde-modified LDL.

[0037] In yet another aspect, the invention concerns a kit forconducting a sandwich assay for the determination of OxLDL orMDA-modified LDL or both in a sample, said kit comprising a substrate onwhich is bound a first antibody that has high affinity for OxLDL orMDA-modified LDL or both, the OxLDL and MDA-modified LDL each having atleast 60 substituted lysine moieties per apo B-100 moiety, and a labeledantibody having a high affinity for OxLDL that becomes bound to thefirst antibody during the assay or for MDA-modified LDL that becomesbound to the first antibody during the assay or for both that becomebound to the first antibody during the assay. Preferably the kit furthercomprises a reactive substance for reaction with the labeled antibody(e.g., an enzyme) to give an indication of the presence of the labeledantibody. Preferably the kit also comprises the stable standards, e.g.,in the form of stable calibrators and/or stable controls. Thus, e.g.,the bound antibody may be mAb-4E6 or mAb-1H11 and the labeled antibodymay be mAb-8A2.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings are provided to help further describethe invention, which drawings are as follows:

[0039]FIG. 1 illustrates the correlation between amounts of oxidized andMDA-modified LDL in coronary lesions in Watanabe heritablehyperlipidemic rabbits (A) and in miniature pigs (B).

[0040]FIG. 2 illustrates the accumulation of OxLDL and MDA-modified LDLin coronary arteries of cardiac explants of ischemic heart disease butnot of dilated cardiomyopathy patients.

[0041]FIG. 3 illustrates the inhibition of the binding of mAb-4E6 toimmobilized OxLDL by native LDL, OxLDL and MDA-modified LDL in solution.

[0042]FIG. 4 illustrates a typical standard curve obtained withMDA-modified LDL in sandwich ELISA.

[0043]FIG. 5 illustrates levels of OxLDL and aldehyde-modified LDL inposttransplant plasma samples of heart transplant patients withdifferent extents of angiographically assessed coronary artery stenosis.

[0044]FIG. 6 illustrates the correlation between plasma levels of OxLDLand aldehyde-modified LDL and titers of specific autoantibodies.

[0045]FIG. 7 illustrates the correlation between plasma levels of OxLDLand aldehyde-modified LDL and of von Willebrand factor antigen.

[0046] These drawings are provided for illustrative purposes only andshould not be used to unduly limit the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention will be further described in conjunctionwith the following examples, which are for illustrative purposes andwhich should not be used to unduly limit the invention.

EXAMPLES Example 1

[0048] Preparation and Characterization of Antibodies Specific for OxLDLand for Aldehyde-Modified LDL

[0049] Balb/c mice were immunized by intravenous and intraperitonealinjection of either OxLDL or MDA-modified LDL. OxLDL was obtained by invitro incubation of LDL (final apo B-100 concentration 700 μg/ml) withcopper chloride (final concentration 640 μM) for 16 h at 37° C.MDA-modified LDL was prepared by incubation of LDL (final apo B-100concentration 700 μg/ml) with a 0.25 M MDA-solution for 3 h at 37° C.The numbers of substituted lysines, measured in the TBARS assay, wastypically 210 per apo B-100 molecule for OxLDL and 240 for MDA-modifiedLDL. Hybridomas were obtained by PEG induced fusion of spleenlymphocytes derived from immunized mice with P3-X63/Ag-6.5.3 myelomacells according to standard techniques (4). The screening for hybridomasproducing specific antibodies was performed with ELISA using microtiterplates coated with malondialdehyde-modified LDL or copper-oxidized LDL.308 hybridomas were obtained after immunization of mice with eitherOxLDL (211) or MDA-modified LDL (97). Hyb4E6 produced antibodiesspecific for both malondialdehyde-modified and copper-oxidized LDL(mAb-4E6), and Hyb1H11 produced antibodies specific formalondialdehyde-modified LDL (mAb-1H11) alone. The IgG fraction of theantibodies was purified by affinity chromatography on proteinA-Sepharose and the affinity of the purified IgGs was determined in asolid phase radioimmunoassay and/or in ELISA. The K_(a) values of themonoclonal antibody mAb-4E6 were <10⁶ M⁻¹ for native LDL and >10⁹ M⁻¹for malondialdehyde-modified LDL and copper-oxidized LDL. The K_(a)values for the monoclonal antibody mAb-1H11 were <10⁶ M⁻¹ for both LDLand OxLDL and >10⁹ M⁻¹ for malondialdehyde-modified LDL. The K_(a)values for the monoclonal antibody mAb-8A2, obtained after immunizationof mice with LDL, were >10⁹ M⁻¹ for all LDL forms. Delipidation ofMDA-modified LDL and OxLDL resulted in a loss of the immunoreactivity ofmAb-4E6, suggesting that it is directed against a conformational epitopein the protein moiety of oxidatively modified LDL.

Example 2

[0050] Use of mAb-4E6 for the Quantitation of OxLDL and MDA-Modified LDLin Coronary Lesions of Watanabe Heritable Hyperlipidemic Rabbits andMiniature Pigs on a Cholesterol Rich Diet

[0051] Coronary arteries were obtained from 2 and 5 month old Watanabeheritable hyperlipidemic rabbits (n=30) on normal chow or from miniaturepigs (n=26) which were fed a diet enriched in cholesterol (4%),saturated fat (14% beef tallow) and bile extract (1%) for 6 to 24 weeks.

[0052] Arterial specimens were submerged within 30 min after removal inPBS (pH 7.4) containing 4% sucrose, 20 μM vitamin E and 10 μM butylatedhydroxytoluene as antioxidants, and 1 mM EDTA, snap-frozen in liquidnitrogen and stored at −80° C. Frozen 7 μM sections were stained withhematoxylin and eosin and with oil red 0 or immunostained as describedbelow. Morphometric parameters of atherosclerotic lesions were measuredby planimetry using the Leica 2 Quantimet color image analyzer(Cambridge, UK). The area within the external elastic lamina, theinternal clastic lamina and the lumen were measured. Media was definedas the area between the internal and external clastic lamina. Intima wasdefined as the area within the internal elastic lamina not occupied byvessel lumen.

[0053] Oxidized apo B-100 containing lipoproteins were detected with thespecific monoclonal antibody mAb-4E6, alkaline-phosphatase conjugatedrabbit-anti-mouse IgG antibodies and the fuchsin alkaline phosphatesubstrate system (Dako, Carpinteria, Calif.), and the absorbance wasmeasured in the color image analyzer. Specificity of immunostaining wasconfirmed by inhibition of staining with excess of copper-oxidized LDLbut not with native LDL or with malondialdehyde-modified albumin. Thestaining co-localized with that monoclonal antibody mAb-13F6, specificfor apo B-100. Absorbance (approximately 10%) measured with excesscopper-oxidized LDL was presumed to represent background staining.

[0054]FIG. 1 illustrates the correlation between the levels of oxidizedapo B-100 containing lipoproteins, i.e. OxLDL and MDA-modified LDL, inthe lesions and the mean intimal area of coronary lesions in Watanabehyperlipidemic rabbits (A) and in miniature pigs (B). Those data thusdemonstrate a correlation between the accumulation of OxLDL andMDA-modified LDL and the progression of coronary atherosclerotic lesionsin 2 different animal models. In Watanabe rabbits the progression of thelesions is due to the increase of LDL cholesterol associated with theheritable LDL receptor deficiency, whereas the progression in miniaturepigs is due to a diet-induced increase in LDL cholesterol.

Example 3

[0055] 5 Immunohistochemistry

[0056] 1. Introduction

[0057] This example is a typical example of the use of the highlyspecific antibody mAb-4E6 in immunohistochemistry applied to humanatherosclerotic lesions. In a similar manner corresponding experimentsmay be performed, for which certain conditions can be adapted by theskilled person using his common knowledge in the field.

[0058] 2. Material and Methods

[0059] Coronary artery specimens, obtained at the time oftransplantation from patients with ischemic heart disease (n=7) ordilated cardiomyopathy (n=7), were treated as described earlier(document 7). The specimens were collected within 30 min after removalof the heart in PBS (pH 7.4) containing 4% sucrose, 20 μM vitamin E and10 μM butylated hydroxytoluene as antioxidants, and 1 mM EDTA, and werestored at −80° C. Frozen 7 μm thick sections were cut and stained withhematoxylin and eosin. Six to 8 sections at a distance of 84 μm wereanalyzed for each specimen to insure representative results. Duplicateslides were developed with monoclonal antibodies mAb-4E6, specific foroxidized LDL, PG-M1, specific for human macrophages, or 1A4, specificfor human smooth muscle α-actin (both from Dako SA, Glostrup, Denmark).Specificity of binding of mAb-4E6 was confirmed by its inhibition withOxLDL but not with native LDL.

[0060] 3. Results

[0061] Coronary artery segments of 7 individuals with pretransplantdilated cardiomyopathy did not contain atherosclerotic lesions and themonoclonal antibody did not detect OxLDL and/or aldehyde-modified LDL inthese segments. Coronary artery segments of 7 patients withpretransplant ischemic heart disease all contained atheroscleroticlesions which contained OxLDL and/or aldehyde-modified LDL (FIG. 2).This information is sufficient to state that the antibody detects OxLDLin atherosclerotic lesions in a highly specific manner.

[0062] OxLDL was associated with macrophage foam cells (preferentiallyin lesions with <50% stenosis), with smooth muscle foam cells and withthe necrotic lipid core (preferentially in lesions with >50% stenosis).Macrophages and smooth muscle cells were identified by immunostainingwith specific monoclonal antibodies (5). These data supported thehypothesis that oxidation of LDL may be associated with the developmentof ischemic coronary artery disease. The monoclonal antibody mAb-4E6 ofthe present invention that detected the immunoreactive material in thetissue sections was then further used in ELISA (cf. Example 4).

[0063] 4. Legend to FIG. 2

[0064] Light micrographs (a, c, e; ×40) and phase contrast micrographs(b, d, f; ×400) of representative left anterior descending coronaryartery specimens of a patient with dilated cardiomyopathy (male; 40years of age) (a, b) and of a patient with ischemic heart disease (male;57 years of age) (c-f). Tissue sections were immunostained with themonoclonal antibody mAb-4E6. Oxidized LDL was undetectable in theneointima of the first patient (a, b), but demonstrable in plaques ofthe second patient. The oxidized LDL was associated with macrophage foamcells that infiltrated at the shoulder areas of fibrous plaques (c, d)and with smooth muscle foam cells in fibrous caps (e, f).

Example 4

[0065] Competitive ELISA

[0066] 1. Introduction

[0067] According to the invention an ELISA was established for thequantitation of OxLDL and aldehyde-modified LDL in plasma. It was basedon the inhibition of the binding of mAb-4E6 to the wells of microtiterplates coated with copper-oxidized LDL. This antibody was obtained asdescribed in Example 1.

[0068] 2. Material and Methods

[0069] Standard OxLDL and aldehyde-modified LDL and plasma samples werediluted in PBS containing 1 mM EDTA, 20 μM vitamin E, 10 μM butylatedhydroxytoluene, 20 μM dipyridamole and 15 mM theophylline to prevent invitro LDL oxidation and platelet activation. Equal volumes of dilutedpurified mAb-4E6 solution (final concentration 7.5 ng/ml) and of dilutedstandard solution (copper-oxidized LDL added as competing ligand at afinal concentration ranging from 50 to 500 ng/ml) were mixed andincubated for 30 min at room temperature. Then 200 μl aliquots of themixtures were added to wells coated with MDA-modified LDL or OxLDL.

[0070] Samples were incubated for 2 h at room temperature. Afterwashing, the wells were incubated for 1 h with horse-radish peroxidaseconjugated rabbit IgG raised against mouse immunoglobulins and washedagain. The peroxidase reaction was performed as described earlier (5)and the absorbance (A) was read at 492 nm.

[0071] Controls without competing ligand and blanks without antibodywere included routinely. The percent inhibition of binding of mAb-4E6 tothe immobilized ligand was calculated as:$\frac{{A^{492{nm}}{control}} - {A^{492{nm}}{sample}}}{{A^{492{nm}}{control}} - {A^{492{nm}}{blank}}}$

[0072] and standard curves were obtained by plotting the percentage ofinhibition vs the concentration of competing ligand.

[0073] The lower limit of detection was 0.020 mg/dl in undiluted humanplasma. Intra- and interassay coefficients of variation were 10 and 12%,respectively. Standard OxLDL and aldehyde-modified LDL and plasmasamples were diluted in PBS containing antioxidants and antiplateletagents as described above.

[0074] 3. Results

[0075] The specificity of mAb-4E6 for OxLDL and aldehyde-modified LDL isillustrated in FIG. 3. 50% inhibition of binding of mAb-4E6 toimmobilized OxLDL and aldehyde-modified LDL was obtained with 0.025mg/dl copper-oxidized LDL and 25 mg/dl native LDL, respectively. The C₅₀value, i.e., the concentration that is required to obtain 50% inhibitionof antibody binding, increased from 2.5 mg/dl for MDA-modified LDL with60 substituted lysine residues per apo B-100 molecule to 0.025 mg/dl forMDA-modified LDL with 240 substituted lysine residues per apo B-100molecule (FIG. 3). Copper-oxidation resulted in fragmentation of the apoB-100 moiety but did not abolish the binding of mAb-4E6 (FIG. 3).50-fold higher molar concentrations of MDA-modified albumin wererequired to obtain 50% inhibition (not shown), whereas up to 1,000-foldhigher molar concentrations of MDA-modified lysine did not affectmAb-4E6 binding. OxLDL and aldehyde-modified LDL isolated from patientplasma had the same reactivity as MDA-modified LDL with 120 substitutedlysines and as copper-oxidized LDL with 210 substituted lysines. Intra-and interassay coefficients of variation were 10 and 12%, respectively.When copper-oxidized LDL were added to human plasma at a finalconcentration of 0.25 and 2 mg/dl, respectively, recoveries were 95 and105%, respectively.

[0076] 4. Legend to FIG. 3

[0077] Interaction of mAb-4E6 with competing ligands in solution.Copper-oxidized LDL (1 μg/ml) was the plated antigen. mAb-4E6 was addedin the absence and in the presence of competing ligands: copper-oxidizedLDL (∇), MDA-modified LDL with 240 (▪), 120 (⋄), 90 (◯) and 60 ()blocked or substituted or modified lysines per apo B-100, respectively,native LDL (Δ), and OxLDL and aldehyde-modified LDL (♦) isolated fromthe plasma of severe chronic renal failure patients. Results areexpressed as B/B₀ where B₀ is the amount of mAb-4E6 bound in the absenceand B that amount bound in the presence of competing ligand.

Example 5

[0078] Sandwich ELISA

[0079] 1. Introduction

[0080] According to the invention a sandwich-type ELISA was establishedfor the quantitation of OxLDL and aldehyde-modified LDL in plasma. Itwas based on the binding of immunoreactive material to the wells ofmicrotiter plates coated with the monoclonal antibody mAb-4E6 and thedetection of bound immunoreactive material with the use of themonoclonal antibody mAb-8A2 labeled with peroxidase. This version of theELISA is more suited for use in the clinical laboratory because itovercomes the need to prepare standard solutions of in vitro oxidizedand/or aldehyde-modified LDL which can only be kept at −4° C. for alimited period of time, typically 2 weeks. MDA-modified LDL may be addedto reference plasma and those standard preparations may be stored at−80° C. for up to 1 year (see above).

[0081] 2. Material and Methods

[0082] Standard preparations and plasma samples were diluted in PBScontaining antioxidants and antiplatelet agents as described above, 180μl aliquots of 80-fold diluted plasma and of standard solutionscontaining between 10 and 0.01 nM of malondialdehyde-modified LDL wereapplied to the wells of microtiter plates coated with mAb-4E6 (200 μl ofa 4 μg/ml IgG solution) and incubated for 2 h at room temperature. Afterwashing, the wells were incubated for 1 h with horseradish peroxidaseconjugated mAb-8A2, IgG (final IgG concentration 65 ng/ml) and washedagain. The peroxidase reaction was performed as described above. Theabsorbance measured at 492 nm correlates with the log-value of thealdehyde-modified LDL concentration in the range between 1.5 nM and 0.3nM.

[0083] 3. Legend to FIG. 4

[0084] Standard curves for the sandwich ELISA mAb-4E6 was the platedantibody. MDA-modified LDL was the ligand. Bound MDA-modified LDL wasdetected with mAb-8A2 conjugated to horse radish peroxidase.MDA-modified LDL was added to 8 different plasma samples to a finalconcentration of 100 nM and further diluted in buffer to finalconcentrations ranging from 2 to 0.2 nM.

Example 6

[0085] Use of the ELISA in Diagnosis of Posttransplant Coronary ArteryDisease

[0086] 1. Introduction

[0087] The ELISA of the present invention was used to study theassociation between plasma levels of OxLDL and aldehyde-modified LDL andposttransplant coronary artery disease.

[0088] 2. Material and Methods

[0089] 2.1. Patients

[0090] The posttransplant study group contained 47 patients transplantedfor dilated cardiomyopathy and 60 patients treated for ischemic heartdisease. The clinical characteristics of these patients are summarizedin Table 1. At the time of blood sampling, between 12 and 84 monthsafter surgery, all patients were in a stable cardiac condition withoutevidence of acute rejection. From 14 patients (7 dilated cardiomyopathyand 7 ischemic heart disease patients) coronary arteries of cardiacexplants were isolated and studied by immunohistochemistry (asdemonstrated in Example 3). Adequate information about smoking habitswas available for 92 of the 107 patients (16 smokers and 76non-smokers). There was no adequate information about smoking habits ofdonors. Blood samples of 53 non-smoking controls (25 males/28 females;age: 52±1.3 years) without a history of atherosclerotic cardiovasculardisease were obtained. The controls were matched for age, gender andlevels of LDL cholesterol. They were selected from the laboratory andclinical staff.

[0091] 2.2. Coronary Angiography

[0092] Routine annual coronary angiograms were available for allposttransplant patients at the time of blood sampling. Coronary arterydisease was independently assessed by two angiographers who whereunaware of the OxLDL and aldehyde-modified LDL levels and was visuallygraded as follows:

[0093] grade 0: normal coronary arteries

[0094] grade I: minor abnormalities with <50% stenosis of primary orsecondary branches and normal left ventricular function

[0095] grade II: ≧50% stenosis of primary or secondary branches, ordistal involvement with impaired left ventricular function.

[0096] It is well known that angiography systematically underestimatesthe extent of coronary intimal thickening in cardiac transplantrecipients. This study therefore does not attempt to accurately quantifythe coronary artery disease in our patients. Rather the subdivision ingroups defined above relies on angiographic data that are easilydistinguishable and that have been shown to correlate withhistopathologic findings. Out of 107 patients, 46 patients had a normalcoronary angiogram 3 years before and development of angiographic,coronary artery disease within a 3 year follow-up period was assessed inall these patients. The reference normal coronary angiogram was thefirst post-operative angiogram in 18 patients, the second in 14 patientsand the third in 14 patients.

[0097] The study was approved by the Institutional Review Board and thestudy subjects provided informed consent.

[0098] 2.3. Blood Sampling

[0099] Venous blood samples from patients and controls were collected on0.1 vol of 0.1 M citrate, containing 1 mM EDTA, 20 μM vitamin E, 10 μMbutylated hydroxytoluene, 20 μM dipyridamole and 15 mM theophylline toprevent in vitro LDL oxidation and platelet activation. Blood sampleswere centrifuged at 3,000 g for 15 min at room temperature within 1 h ofcollection and stored at −20° C. until the assays were performed.

[0100] 2.4. Lipoproteins: Isolation and Modification

[0101] LDL were isolated from pooled sera of fasting normolipidemicdonors by density gradient ultracentrifugation (document 6). Standardpreparations of MDA-modified and copper-oxidized LDL were prepared asdescribed elsewhere (7, 8) and were used as assay controls. Apo B-100molecules of in vitro MDA-modified LDL (7) and of copper-oxidized LDL(8) contained on average 244, and 210 substituted lysines (out of atotal of 356), respectively (5, 9). Whereas the extent oflysine-substitution in in vitro MDA-modified LDL and copper-oxidized LDLis very similar, the lipid moiety of the former is not oxidized.Specificity of the monoclonal antibody mAb-4E6 for both MDA-modified LDLand copper-oxidized LDL suggests that it depends on the extent ofprotein (lysine) modification only. All lipoprotein concentrations weretherefore expressed in terms of protein. OxLDL and aldehyde-modified LDLisolated from the plasma of patients were characterized as describedpreviously (5, 9).

[0102] 2.5. Assays

[0103] Cholesterol and triglycerides were measured by enzymatic methods(Boehringer Mannheim, Meylon, France). Typing of majorhistocompatibility complex class I (HLA-B) and class II (HLA-DR) antigenwas performed by the microlymphocytotoxicity technique.

[0104] The ELISA of the invention was used to detect OxLDL andaldehyde-modified LDL.

[0105] 2.6. Statistical Analysis

[0106] Controls and patients were compared by ANOVA test followed bynonparameteric Mann-Whitney or Dunnett's multiple comparison test onlogarithmically transformed values, in the Instat V2.05a statisticalprogram (Graph Pad Software, San Diego, Calif.). Non-quantitativeparameters were compared by Chi-square analysis. OxLDL andaldehyde-modified LDL levels measured in 3 aliquots of the same plasmasamples were compared in Friedman nonparametric repeated measures test.Logistic regression analysis, using the SAS software (SAS InstituteInc., USA), was performed to evaluate the correlation betweenangiographically assessed coronary artery stenosis (as dependentvariable) and plasma levels of OxLDL and aldehyde-modified LDL, age andsex of recipients, age and sex of donors, pretransplant history ofischemic heart disease or dilated cardiomyopathy, duration of ischemia,length of follow up, number of rejections, number of HLA-mismatches,cytomegalovirus infection, hypertension (antihypertensive treatment),diabetes, treatment with lipid lowering drugs (statins or fibrates) andserum levels of LDL cholesterol, HDL cholesterol and triglycerides asindependent variables. p-values of less than 0.05 were considered toindicate statistical significance. Logistic regression analysis was alsoperformed to evaluate the correlation between plasma levels of OxLDL andaldehyde-modified LDL and development of coronary artery stenosis duringa 3-year follow-up period.

[0107] 3. Results

[0108] The correlation between OxLDL and aldehyde-modified LDL andcoronary artery stenosis was evaluated in 47 patients transplanted fordilated cardiomyopathy and in 60 patients treated for ischemic heartdisease. Analysis of clinical data for the two groups of hearttransplant patients (Table 1) revealed no significant differences in ageand gender of the recipients, age and gender of donors, duration ofischemia of the donor heart, number of rejection episodes, number ofHLA-mismatches, frequency of Cytomegalovirus infections, hypertension ordiabetes, and grade of coronary artery stenosis. Patients transplantedfor ischemic heart disease were followed longer and received morefrequently lipid lowering drugs (Table 1).

[0109] Analysis of the laboratory data (Table 2) revealed no significantdifferences in serum levels of triglycerides, HDL cholesterol and LDLcholesterol between groups of patients or between patients and controls.However, significant differences in levels of OxLDL andaldehyde-modified LDL were observed. Mean plasma levels of OxLDL andaldehyde-modified LDL were 1.3±0.14 mg/dl in dilated cardiomyopathypatients (p<0.001 vs controls) and 1.7±0.13 mg/dl in ischemic heartdisease patients (p<0.001 vs controls and <0.01 vs dilatedcardiomyopathy patients) (Table 2). Plasma levels of OxLDL andaldehyde-modified LDL in control subjects matched for age, gender andserum levels of triglycerides, HDL cholesterol and LDL cholesterol were0.60±0.034 mg/dl (n=53; p<0.001 vs both transplanted dilatedcardiomyopathy and ischemic heart disease patients).

[0110] Levels of OxLDL and aldehyde-modified LDL were not different insamples that were stored for 24 h to 4 months after collection, and upto four thawing and freezing cycles did not cause an increase of OxLDLand aldehyde-modified LDL levels. These findings indicated that theaddition of EDTA, antioxidants and anti-platelet agents adequatelyprevented the in vitro oxidation of LDL. In a subset of 87 consecutiveplasma samples levels of OxLDL and/or aldehyde-modified LDL weremeasured in 3 separate aliquots on 3 different days. The levels were1.30±0.074 mg/dl, 1.48±0.101 mg/dl and 1.46±0.090 mg/dl, respectively.Friedman nonparametric repeated measures test revealed no significantdifferences.

[0111] Mean OxLDL and aldehyde-modified LDL levels were 1.2±0.053 mg/dl(n=79) in posttransplant samples of patients with angiographicallynormal coronary arteries (grade 0), 2.1±0.30 mg/dl in patients withgrade I coronary artery stenosis (n=18; p<0.001 vs grade 0) and 3.2±0.45mg/dl in patients with grade II coronary artery stenosis (n=10; p<0.001vs grade 0 and p<0.05 vs grade I) (FIG. 5). Serum levels of LDLcholesterol, triglycerides and HDL cholesterol were very similar inpatients with higher grade of coronary artery stenosis. Levels of OxLDLand aldehyde-modified LDL in plasma samples of patients transplanted fordilated cardiomyopathy or ischemic heart disease, with the same grade ofcoronary artery stenosis, were similar: 1.1±0.072 and 1.4±0.079 mg/dlfor grade 0 patients and 2.6±0.60 and 2.4±0.29 mg/dl for patients withhigher grade of coronary artery stenosis. The number of patients withelevated levels of OxLDL and aldehyde-modified LDL (>1 mg/dl, i.e. meanlevels of controls+2 SD) were 43 (out of 60) in the subpopulation ofpatients with pretransplant ischemic heart disease and 21 (out of 47) inthe subpopulation of patients with pretransplant dilated cardiomyopathy.Forty-two out of 79 patients with angiographically normal coronaryarteries had elevated levels of OxLDL and aldehyde-modified LDL.Elevated levels were detected in 12 (out of 18) patients with grade Iand in all patients with grade II stenosis (p=0.0046 for trend).

[0112] To allow further characterization of the immunoreactive materialdetected in the ELISA, LDL fractions were isolated from the plasma ofall of 10 patients with grade II coronary artery stenosis (18). Thesefractions retained 85±10% (mean±SD) of the immunoreactive material,whereas no immunoreactive material migrated in the serum albuminposition. OxLDL and aldehyde-modified LDL were isolated from isolatedLDL fractions by ion-exchange chromatography on a mono Q-Sepharosecolumn with a recovery of 75%. The number of substituted lysines per apoB-100 molecule was 130±10 for OxLDL and aldehyde-modified LDL comparedto 5±1 (p<0.001) for native LDL. The respective cholesterol/proteinratios were 3.3±0.54 and 1.8±0.36 (p<0.001). The levels of arachidonateand linoleate in OxLDL and aldehyde-modified LDL isolated from theplasma of these patients were 75 and 80% lower than these in native LDLisolated from the same plasma samples. The inhibition curves obtainedwith OxLDL and aldehyde-modified LDL isolated from the plasma of hearttransplant patients were superimposable with these obtained with invitro oxidized LDL with the same extent of protein modification (120substituted lysines per apo B-100 molecule) (FIG. 3).

[0113] The protein/antigen ratio and the relative reactivity in theELISA of OxLDL and aldehyde-modified LDL isolated from the plasma ofthese patients were similar to these of copper-oxidized or MDA-modifiedstandard LDL preparations.

[0114] Logistic regression analysis (Table 3) identified 3 parametersthat were significantly and independently correlated with posttransplantcoronary artery stenosis including levels of OxLDL and aldehyde-modifiedLDL, length of follow up and donor age.

[0115] In contrast, pretransplant history of dilated cardiomyopathy orof ischemic heart disease, age and gender of recipients, gender ofdonors, duration of ischemia of the donor heart, extent of HLA-mismatch,number of rejections, hypertension, diabetes, and serum levels of LDLcholesterol, HDL cholesterol and triglycerides in recipients did notsignificantly contribute to the individual variations in extent ofcoronary artery stenosis (Table 3).

[0116] Serum levels of LDL cholesterol, HDL cholesterol andtriglycerides in patients were similar to these in controls (Table 2),so that higher grade of coronary artery stenosis was unlikely to dependon these variables in this study group. Fifty-six of the 107 transplantpatients received lipid lowering drugs (46 with statins and 10 withfibrates) (Table 1), but the treatment with these drugs was notcorrelated with the incidence of angiographic graft vasculopathy (Table3). Seventy-five (out of 107) patients were treated with calcium channelblockers. The plasma levels of OxLDL and aldehyde-modified LDL in thesepatients (1.53±0.11 mg/dl) were very similar to these in non-treatedpatients (1.74±mg/dl) and treatment with these drugs was not correlatedwith the extent of coronary artery stenosis.

[0117] Development of coronary artery disease was observed in 12 out of46 heart transplantation patients during a 3-year follow-up period.There were no differences in age and gender of recipients, age andgender of donors, duration of ischemia, extent of HLA mismatch,frequency of cytomegalovirus infections, hypertension and diabetes(Table 4) nor in serum levels of triglycerides, HDL cholesterol and LDLcholesterol (Table 5) between patients without and with development ofcoronary artery disease. However, levels of OxLDL and aldehyde-modifiedLDL were significantly elevated in patients with development of coronaryartery disease (Table 5).

[0118] Logistic regression analysis revealed that plasma levels of OxLDLand aldehyde-modified LDL (Chi-square value=7.1; p=0.0076) and age ofdonor (Chi-square value=4.4; p=0.035) predicted the development ofcoronary artery disease in these patients. Three of these patientsdeveloped coronary artery disease in the first year, 3 in the second and6 in the third year. The plasma levels of OxLDL and aldehyde-modifiedLDL were 3.9±0.6 mg/dl, 2.0±0.37 mg/dl and 1.2±0.33 mg/dl, respectively.Although statistical analysis showed no correlation with gender,hypertension and Cytomegalovirus infection, 8 out of 12 of thesepatients were male, hypertensive and had Cytomegalovirus infection.

[0119] 4. Discussion

[0120] This demonstrates:

[0121] 1) that cardiac explants of patients with ischemic heart disease,but not with dilated cardiomyopathy, contain oxidized LDL in macrophagesand in smooth muscle cells in atheromatous plaques;

[0122] 2) that posttransplant coronary artery disease is associated withincreased plasma levels of OxLDL and aldehyde-modified LDL both inpatients transplanted for dilated cardiomyopathy or for ischemic heartdisease, and

[0123] 3) that increased plasma levels of OxLDL and aldehyde-modifiedLDL correlate with the development of coronary artery stenosis.

[0124] OxLDL and aldehyde-modified LDL levels in plasma samples of hearttransplant patients without angiographically detectable coronary arterylesions were 2-fold higher than in plasma samples of control subjectswithout a history of atherosclerotic cardiovascular disease, who werematched for age, gender, and plasma levels of LDL cholesterol, HDLcholesterol and triglycerides. A further 2.7-fold increase was observedin posttransplant plasma samples of patients with pronounced coronaryartery stenosis. These data suggest that elevated plasma levels of OxLDLand aldehyde-modified LDL may be an indicator of posttransplant coronaryartery stenosis. Increased plasma levels of OxLDL and aldehyde-modifiedLDL correlated with the extent of coronary artery stenosis and also withits progression, suggesting that OxLDL and aldehyde-modified LDL mayplay a pathogenic role in the accelerated progression of coronary arterydisease in heart transplant patients.

[0125] It has been suggested that posttransplant atherosclerosis resultsfrom a “response to injury” of the endothelium (10). The extent ofischemic injury in endomyocardial biopsies was indeed found to be astrong predictor of the development of accelerated atherosclerosis(11-13). Endothelial injury may be induced by cellular delayed-typehypersensitivity immune responses elicited by class IIhistocompatibility (HLA) antigens on coronary artery endothelium (14),by cytomegalovirus infection (15, 16), by cyclosporin (17) and by OxLDLand aldehyde-modified LDL (18) that may act synergistically withcyclosporin (19). In the present study, the extent ofhistoincompatibility between pairs of donors and recipients, the numberof episodes of rejection or Cytomegalovirus infection did not correlatewith the grade of coronary artery stenosis, whereas OxLDL andaldehyde-modified LDL were significantly and independently correlatedwith posttransplant coronary artery disease. The observed associationbetween the age of the donor and the occurrence of coronary arterydisease is in agreement with previous findings that coronaryatherosclerosis in the donor heart predisposes to acceleratedposttransplant coronary artery stenosis (20).

[0126] OxLDL and aldehyde-modified LDL were demonstrated in coronaryarteries in cardiac explants of ischemic heart disease patientssuggesting that OxLDL and aldehyde-modified LDL that accumulate in thearterial wall may contribute to the progression of coronary arterystenosis. The cholesterol/protein ratio in OxLDL and aldehyde-modifiedLDL was very similar to that in LDL extracted from atheroscleroticlesions as described previously (21,22). A possible explanation is thatat least part of the OxLDL and aldehyde-modified LDL is released fromthe arterial wall. Previously, we have demonstrated that plaque rupturein acute myocardial infarction patients is associated with the releaseof oxidatively modified LDL (5).

[0127] In vitro data suggest that OxLDL and aldehyde-modified LDL may belinked to atherogenesis by a sequence of events (reviewed in 2,23).Endothelial cells exposed to OxLDL and aldehyde-modified LDL secreteadhesion molecules, chemoattractant proteins and colony-stimulatingfactors that enhance the infiltration, proliferation and accumulation ofmonocytes/macrophages in the arterial wall. Uptake of OxLDL andaldehyde-modified LDL by infiltrated macrophages may result in thegeneration of foam cells that produce oxygen radicals and thus furthercontribute to the oxidation of LDL. It has been demonstrated that OxLDLand aldehyde-modified LDL inhibit the migration of aortic endothelialcells in vitro, suggesting that OxLDL and aldehyde-modified LDL maylimit the healing response of the endothelium after injury, and thatbasic fibroblast growth factor reverses the atherosclerosis associatedimpairment of human coronary angiogenesis-like responses in vitro(24,25). OxLDL and aldehyde-modified LDL may also contribute to rapidlyprogressing coronary atherosclerosis by inducing platelet adhesion, bydecreasing the anticoagulant and fibrinolytic capacities of activatedendothelium and by impairing vasodilation and inducing shear stress(2,23).

[0128] Increased intracellular levels of ferritin (26) or ofalpha-tocopherol analogs (27) decreased the extent of endothelial injuryelicited by OxLDL and aldehyde-modified LDL in vitro, whereasantioxidants protect against progression of atherosclerosis inexperimental animals (reviewed in document 28).

[0129] In summary, the present example demonstrates that posttransplantatherosclerosis correlates with plasma levels of OxLDL andaldehyde-modified LDL.

[0130] 5. Legend to the FIG. 5

[0131] Plasma levels of OxLDL and aldehyde-modified LDL andangiographically assessed grade of coronary artery stenosis. Grade 0:normal coronary arteries; grade I: minor abnormalities with <50%stenosis of primary or secondary branches and normal left ventricularfunction; and grade II: ≧50% stenosis of primary or secondary branches,or distal occlusions with impaired left ventricular function.

Example 7

[0132] Use of the ELISA in Renal Failure Patients

[0133] 1. Material and methods

[0134] 1.1. Subjects

[0135] The patient population consisted of 20 mild chronic renal failure(MCRF) and 77 severe chronic renal failure patients: 21 on conservativetreatment including dietary and antihypertensive treatment (SCRF), and56 on a four-hour, three times a week hemodialysis schedule (HEMO) for66 months (95% CI, 50-82 months). All hemodialysis patients were givenan oral polyvitamin preparation (Ol-Amine, La Meuse, Belgium) afterhemodialysis, which contained only minute amounts of antioxidantcompounds (i.e. 5 mg of vitamin E and 100 mg of vitamin C). Controls andnon-dialyzed patients did not receive routine prescriptions of vitaminsupplements. The high frequency of atherosclerotic disease in thesepatients (Table 6) is in agreement with previously published data (29,30). The diagnosis of atherosclerotic heart disease, cerebrovasculardisease and peripheral vascular disease was made after reviewing thepatient files for a history of myocardial infarction, unstable angina orantianginal treatment, cerebrovascular accidents, transient ischemicattack or events related to peripheral vascular disease such as ischemiculcera, amputation or bypass surgery. Angiograms were available for onlya few patients. No patients had evidence of unstable atheroscleroticdisease at the time of blood sampling nor in the following days. A groupof 27 healthy volunteers (Table 6) without a history of renal disease oratherosclerotic vascular disease served as controls. Patients receivinglipid lowering drugs were excluded. The study was approved by theInstitutional Review Board and the study subjects provided informedconsent.

[0136] 1.2. Blood Samples

[0137] Venous blood samples from patients and controls were collected on0.1 vol of 0.1 M citrate, containing 1 mM EDTA, 20 μM vitamin E, 10 μMbutylated hydroxytoluene, 20 μM dipyridamole and 15 mM theophylline toprevent in vitro LDL oxidation and in vitro platelet activation,respectively. Blood samples were centrifuged at 3,000 g for 15 min atroom temperature within 1 h of collection and stored at −20° C. untilthe assays were performed.

[0138] 1.3. Assays

[0139] Titers of autoantibodies against OxLDL and aldehyde-modified LDLand native LDL were measured according to Salonen et al. (3) asdescribed in detail elsewhere (5). vWF antigen levels were measured in asandwich-type ELISA based on a polyclonal rabbit anti-human vWFantiserum (Dako, Glostrup, Denmark), horseradish peroxidase-conjugatedrabbit anti-human vWF IgG (Dako) and o-phenylenediamine. Plasma levelsof total cholesterol, HDL cholesterol and triglycerides were determinedusing standard enzymatic assays (Boehringer Mannheim, Meylon, France).The LDL cholesterol levels were calculated using the Friedewald formula.For the patients not in hemodialysis, creatinine clearance rates werecalculated from plasma creatinine levels using the Cockcroft and Gaultformula (31).

[0140] 1.4. Statistical Analysis

[0141] Controls and patients were compared by ANOVA test followed byDunnett's multiple comparison test, in the Instat V2.05a statisticalprogram (Graph Pad Software, San Diego, Calif.). Correlationcoefficients were calculated according to Spearman. Multiple regressionanalysis, using the SAS software (SAS Institute Inc., USA), wasperformed to study the relationship between OxLDL and aldehyde-modifiedLDL as dependent variable, and age, sex, hypertension (antihypertensivetreatment), levels of triglycerides, HDL cholesterol, LDL cholesteroland creatinine clearance rates (marker of extent of renal failure) andlevels of vWF (marker of endothelial injury) as independent variables.

[0142] 2. Results

[0143] Mean plasma levels of OxLDL and aldehyde-modified LDL in controlswere 0.59 mg/dl (95% CI, 0.52-0.66 mg/dl; n=27), and were 2.7-foldhigher in MCRF patients (p<0.01 as determined by Dunnett's multiplecomparison test), 3.1-fold higher in SCRF patients (p<0.001), and5.4-fold higher in HEMO patients (p<0.001) (Table 7). OxLDL andaldehyde-modified LDL levels were inversely correlated with creatinineclearance rates (r=−0.65; p<0.001; n=73). HEMO patients were notincluded in this analysis because their plasma creatinine clearancecannot be determined adequately.

[0144] In a series of 14 hemodialyzed patients, levels of OxLDL andaldehyde-modified LDL were found to be very similar in fresh and infresh frozen plasma samples. Three freezing and thawing cycles did notcause an increase of OxLDL and aldehyde-modified LDL, indicating thataddition of antioxidants and antiplatelet agents prevented in vitrooxidation.

[0145] Plasma samples were obtained from 14 hemodialyzed patients on 3consecutive days before the start of the dialysis procedure. The levelsof OxLDL and aldehyde-modified LDL in these samples were similar:3.4±0.25 mg/dl, 3.2±0.21 mg/dl and 3.5±0.28 mg/dl, respectively.Furthermore, plasma samples were obtained during (after 2 h) and at theend (after 4 h) of hemodialysis. Plasma levels of OxLDL andaldehyde-modified LDL were 4.0±0.60 mg/dl and 4.7±0.70 mg/dl (p=NS vsbefore) as compared to 3.4±0.25 mg/dl before the start of the dialysisprocedure. Thus the hemodialysis procedure did not induce a significantincrease in the OxLDL and aldehyde-modified LDL levels.

[0146] Adequate information about smoking habits was only available forcontrols (27 non-smokers) and for HEMO patients (12 smokers and 45non-smokers). Levels of OxLDL and aldehyde-modified LDL were somewhathigher in smoking HEMO patients (3.6 mg/dl; 95% CI, 2.1-5.6 mg/dl) thanin non-smoking HEMO patients (3.0 mg/dl; 95% CI, 2.5-3.6 mg/dl; p=NS).The plasma levels of OxLDL and aldehyde-modified LDL in hemodialyzedpatients with a history of unstable atherosclerotic cardiovasculardisease were 3.5±0.40 mg/dl (n=30) as compared to 2.8±0.60 mg/dl (n=26,p=NS) in hemodialyzed patients without a history of unstableatherosclerotic cardiovascular disease.

[0147] LDL fractions were isolated from the plasma of 10 controls, of 10MCRF patients, of 10 SCRF-patients and of 10 HEMO patients by gelfiltration on a Superose 6HR 10/30 column, as described previously (5).75±6% (mean±SD), 80±4%, 83±6% and 79±5% of the immunoreactive materialwas recovered in the LDL fractions. No immunoreactive material migratedin the serum albumin position. The inhibition curves obtained with therespective LDL fractions were parallel to those obtained with in vitrocopper-oxidized or MDA-modified standard LDL preparations. OxLDL andaldehyde-modified LDL were isolated from isolated LDL fractions of 10SCRF patients by ion-exchange chromatography on a mono Q-Sepharosecolumn with a recovery of 75%. Their physicochemical properties aresummarized in Table 8. The levels of arachidonate of OxLDL andaldehyde-modified LDL isolated from these patients were reduced with.75%, whereas its linoleate levels were reduced with 80%. Thirty-seven %of the lysine residues of OxLDL were substituted with aldehydes. Theinhibition curves obtained with OxLDL and aldehyde-modified LDL isolatedfrom the plasma of chronic renal failure patients were parallel to theseobtained with OxLDL and aldehyde-modified LDL that was obtained by invitro oxidation of LDL that had been isolated from the plasma of controlsubjects (FIG. 3). The protein/antigen ratio and the relative reactivityin the ELISA of OxLDL and aldehyde-modified LDL isolated from the plasmaof these patients were similar to these of copper-oxidized orMDA-modified standard LDL preparations (Table 8).

[0148] Titers of autoantibodies against OxLDL and aldehyde-modified LDLwere 4.2 (95% CI, 4.0-4.4) in controls, were similar in MCRF and SCRFpatients, but significantly increased in HEMO patients (p<0.001) (Table7). Autoantibody titers correlated with levels of OxLDL andaldehyde-modified LDL in SCRF patients (r=0.44; p=0.047) and in HEMOpatients (r=0.37; p=0.0055) (FIG. 6). No circulating autoantibodiesagainst native LDL could be detected.

[0149] Levels of vWF were 100 percent in controls (95% CI, 90-110percent), and were 1.5-fold higher in MCRF patients (p=NS vs controls),1.6-fold higher in SCRF patients (p<0.01) and 2.1-fold higher (p<0.001)in HEMO patients (Table 7). Levels of vWF were not significantly higherin smoking HEMO patients (250 percent; 95%, 150-340 percent; n=12) thanin non-smoking HEMO patients (220 percent; 95% CI, 190-260 percent;n=45). Levels of vWF correlated with levels of OxLDL andaldehyde-modified LDL in MCRF patients (r=0.59; p<0.0057), in SCRFpatients (r=0.69; p=0.0006) and in HEMO patients (r=0.62; p<0.0001)(FIG. 7). In contrast, levels of vWF did not correlate with LDLcholesterol levels or with body weight.

[0150] Multiple regression analysis revealed that the extent of renalfailure (F=14; p=0.0004) and the extent of endothelial injury (F=26;p=0.0001), but not age, sex, hypertension, triglyceride levels, HDLcholesterol or LDL cholesterol levels, accounted for a significantfraction of the variations in OxLDL and aldehyde-modified LDL levels(Table 9). Even when only subjects without evidence of ischemicatherosclerotic disease (n=53) were included in the model(R²-value=0.68) only the extent of renal failure (F=21; p=0.0001) andthe extent of endothelial injury (F=14; p=0.0006) contributedsignificantly to the variations in OxLDL and aldehyde-modified LDLlevels. No other variables contributed significantly to these variationsafter exclusion of subjects without evidence of ischemic atheroscleroticdisease. When only subjects with evidence of ischemic atheroscleroticdisease (n=15) were included only the extent of endothelial injury(F=6.2; p=0.047; R²-value=0.65) contributed to the variations in OxLDLand aldehyde-modified LDL levels. Exclusion of diabetic patients did notsignificantly change the data either. After exclusion of the extent ofrenal failure as an independent variable, multiple regression analysisrevealed that hemodialysis (F=5.6; p=0.021; n=77), LDL cholesterollevels (F=7.1; p=0.0095) and endothelial injury (F=35; p=0.0001)accounted for a significant fraction of the variation in OxLDL andaldehyde-modified LDL levels in severe chronic renal failure patients(Table 10).

[0151] 3. Discussion

[0152] In vitro work and experimental animal data suggest that oxidizedLDL (OxLDL and aldehyde-modified LDL) may contribute to the progressionof atherosclerosis (reviewed in document 2), and OxLDL andaldehyde-modified LDL have been demonstrated in human atheroscleroticplaques (5). The immuno-assay of this invention identifies OxLDL andaldehyde-modified LDL (MDA-modified LDL) with ≧60 substituted lysinesper apo B-100 molecule, which represents the threshold of substitutionrequired for scavenger receptor mediated uptake (1). Increased levels ofOxLDL and aldehyde-modified LDL have been measured by ELISA in theplasma of chronic renal failure patients.

[0153] Overall, 80 percent of the immunoreactive material isolated fromthe plasma of patients was recovered in the LDL fractions that wereseparated by gel filtration. No immunoreactive material migrated in thealbumin position. Inhibition curves obtained with the isolated OxLDL andaldehyde-modified LDL were parallel to these of in vitro copper-oxidizedor MDA-modified LDL standard preparations and the protein/antigen ratioand the C₅₀ value of the isolated OxLDL and aldehyde-modified LDL wereidentical to these of standard OxLDL and aldehyde-modified LDLpreparations. These data suggested that increased immunoreactivity ofOxLDL and aldehyde-modified LDL fractions in plasma of these patientswith the antibodies of this invention depended indeed on the higherextent of protein modification and not on changes in lipid compositionas was previously observed with other antibodies (32). The increasedelectrophoretic mobility, the increased lysine modification, theincreased cholesterol/protein ratio, the decreased arachidonic acid andlinoleate levels were very similar to these of modified LDL extractedfrom atherosclerotic lesions (21, 22). OxLDL and aldehyde-modified LDLinduced foam cell generation, suggesting that OxLDL andaldehyde-modified LDL were not “minimally modified” LDL.

[0154] Multiple regression analysis revealed that chronic renal failureand endothelial injury contributed significantly to the variation inOxLDL and aldehyde-modified LDL levels even when patients with evidenceof ischemic atherosclerotic disease were excluded. Indeed, 79.6% and82.4% of the variations in OxLDL and aldehyde-modified LDL levels couldbe explained in these models. No patients had evidence of unstableatherosclerotic disease at the time of blood sampling nor in thefollowing days and exclusion of patients with a history of ischemicatherosclerotic disease did not affect the contribution of the extent ofrenal failure and of endothelial injury to the variations in OxLDL andaldehyde-modified LDL.

[0155] LDL cholesterol levels in controls and patients were very similarand LDL cholesterol levels did not contribute to the variations in OxLDLand aldehyde-modified LDL levels. Sutherland et al. (33) demonstratedthat the lag time of conjugated diene formation, which is a measure forthe sensitivity of LDL to in vitro oxidation, was similar in patientswith chronic renal failure and in matched controls. The maximum rate andthe extent of LDL oxidation were even lower in patients with renaldisease than in controls, due to lower levels of linoleic acid andhigher levels of oleic acid. Furthermore, Schulz et al. (34)demonstrated that despite the fact that hemodialysis causes leukocyteactivation, the in vitro LDL oxidation lag time was similar in renalpatients and in healthy controls. It was concluded that theantioxidative defense of lipoproteins was preserved in renal failure andduring dialysis.

[0156] In experimental models, antioxidants such as probucol and vitaminE were found to protect against glomeral injury (35, 36) and to slowatherogenic processes (28). Renal vasoconstriction caused by cholesterolfeeding was corrected by probucol or by a thromboxane antagonist (35).Galle et al. (38) demonstrated that the inhibition ofendothelium-dependent dilation induced by oxidized lipoprotein could beprevented by high density lipoproteins that are significantly decreasedin hemodialyzed patients. In addition, minerals like selenium andnutrients such as coenzyme Q10 may minimize free radical generation andthus oxidative stress. Folic acid, vitamin B12 and vitamin B6 may beessential in the prevention of hyperhomocysteinemia that may contributeto the endothelial injury (39) and to oxidation of LDL (40) in thesepatients. A diet rich in mono-unsaturated fatty acids (oleic acid,resistant to oxidation) reduced the extent of endothelial injury indiabetes patients (41). Thus it is possible that dietary orpharmacological means may reduce OxLDL and aldehyde-modified LDL and vonWillebrand factor in chronic renal failure and alleviate the enhancedgeneralized atherosclerosis in such patients.

[0157] After adjustment for the extent of renal failure, multipleregression analysis revealed that both LDL cholesterol levels andendothelial injury strongly contributed to the variations in OxLDL andaldehyde-modified LDL levels in severe chronic renal failure patients.

[0158] Hemodialysis results in platelet and leukocyte activation (42,43), which generates oxygen radicals and aldehydes that may alsocontribute to oxidation of LDL. OxLDL and aldehyde-modified LDL may thencontribute to thrombogenesis and atherogenesis by stimulating platelets(44). Because of the rather limited number of patients, subgroupanalysis to further study the interaction between hemodialysis,oxidation of LDL and ischemic atherosclerotic disease could not beperformed (45).

[0159] 4. Legend to FIGS. 6 and 7

[0160]FIG. 6. Correlation between plasma levels of OxLDL andaldehyde-modified LDL (log values) and titers of autoantibodies (logvalues): regression line for severe chronic renal failure patients,either on conservative treatment (Δ; - - - ) (r=0.44; p=0.047) or onhemodialysis (□; - - - ) (r=0.37; p=0.0055). No significant correlationwas observed in controls and in mild chronic renal failure patients.

[0161]FIG. 7. Correlation between plasma levels of OxLDL andaldehyde-modified LDL (log values) and of von Willebrand factor antigen(log values): regression line for mild chronic renal failure patients(◯; - - - ) (r=0.59; p=0.0057) or for severe chronic renal failurepatients either on conservative treatment (Δ; - - - ) (r=0.69; p=0.0006)or on hemodialysis (□; - - - ) (r=0.62; p<0.00001). No significantcorrelation was observed in controls.

Example 8

[0162] Preparation of Reference-Standard for Use in Immunological Assays

[0163] 1. Introduction

[0164] According to the invention it has been found that LDL that ismodified by treatment with malondialdehyde (MDA) is highly stable.Furthermore, the extent of modification is highly reproducible. LDLmodified with MDA in a particular ratio has an identical number ofsubstituted lysines and can therefore be used as a reference sample inimmunological assays. This example shows the preparation of thestandard.

[0165] 2. Material and Methods

[0166] MDA-modified LDL was added to control plasma (containinganti-oxidants and anti-platelet compounds and anti-coagulants) to afinal concentration of 100 nM MDA modified apo B-100. Aliquots werefrozen at −80° C. In 6 days were aliquots were thawed, diluted to finalconcentrations ranging from 10 to 0.1 nM MDA-modified apo B-100 andanalyzed in ELISA (4 dilution curves per day).

[0167] 3. Results

[0168] The inter-assay variation coefficients of 10 subsequent sandwichELISA's of this invention using 10 subsequent, independent MDA-modifiedLDL standard preparations of this invention are summarized in Table 11.

[0169] These data show that for concentrations of MDA-modified LDLranging from 10 and 0.01 nM the inter-assay variation ranged from 7.6 to16.9%.

[0170] Abbreviations

[0171] C₅₀: concentration required to obtain 50% inhibition of antibodybinding

[0172] MDA: malondialdehyde

[0173] HEMO: severe chronic renal failure patients on maintenancehemodialysis

[0174] MCRF: mild chronic renal failure patients

[0175] SCRF: severe chronic renal failure patients on conservativetreatment

[0176] OxLDL: oxidized low density lipoproteins. TABLE 1 Clinical dataof heart transplant patients Heart transplant patients Dilated Ischemiccardiomyopathy heart disease Characteristics (n = 47) (n = 60) p-valuesAge of recipient (yr)  54 ± 1.6   55 ± 0.95 *NS Gender of recipient(M/F) 41/6  53/7  *NS Age of donor (yr)  29 ± 1.5  29 ± 1.4 **NS Genderof donor(M/F) 31/16 44/16 *NS Length of follow up (mo)  39 ± 3.1  50 ±2.7 **0.008 Duration of ischemia (min)  130 ± 7.0   140 ± 5.3  **NS Noof HLA mismatches DR  1.5 ± 0.09  1.4 ± 0.08 **NS B + DR  3.1 ± 0.13 3.0 ± 0.13 **NS No of rejection episodes 0.38 ± 0.13 0.25 ± 0.06 **NSCytomegalovirus infection 26 43 *NS Hypertension 37 53 *NS Diabetes 4 3*NS Coronary artery disease Grade 0 39 40 *NS Grade I 5 13 *NS Grade II3 7 *NS Lipid lowering drugs 17 39  *0.004 Statins 13 33  *0.006Fibrates 4 6 *NS Calcium channel blockers 31 47 *NS

[0177] TABLE 2 Laboratory data of controls and heart transplant patientsHeart transplant patients Dilated Ischemic Control scardiomyopathy (DC)heart disease Characteristics (n = 27) (n = 47) p vs control (n = 60) ps control p-valuesvs DC Serum triglyderides (mg/dl)† 130 ± 11  130 ±8.3  NS 140 ± 7.0  NS NS HDL cholesterol (mg/dl)‡  44 ± 2.1  54 ± 2.5 NS 49 ± 1.9 NS NS LDL cholesterol (mg/dl)‡ 120 ± 4.7  100 ± 4.4  NS 110 ±3.3  NS NS Oxidized LDL (mg/dl)  0.59 ± 0.036  1.3 ± 0.14 <0.001  1.7 ±0.13 <0.001 <0.01

[0178] TABLE 3 Logistic regression analysis of the relation betweenclinical- laboratory data and extent ol coronary artery stenosis inheart transplant patients. Independent variable Chi-square value p-valueOxidised LDL 1.8 0.0001 Length of follow up 1.1 0.0008 Age of donor 3.90.047 Age of recipient 0.12 0.73 Sex of recipient 1.8 0.18 Sex of donor0.025 0.88 History of pretransplant dilated 0.0018 0.97 cardiomyopathy(n = 47) or ischemic heart disease (n = 60) Duration of ischemia 0.250.62 No of HLA mismatches 1.6 0.20 No of rejection episodes 3.0 0.081Cytomegalovirus infection 0.17 0.47 Hypertension 1.9 0.16 Diabetes0.0016 0.97 Treatment with lipid lowering drugs Statins 1.1 0.30Fibrates 0.12 0.73 Treatment with calcium channel blockers 0.16 0.49Serum triglycerides 0.18 0.67 Serum HDL cholesterol 0.25 0.61 Serum LDLcholesterol 0.044 0.83

[0179] TABLE 4 Clinical data of heart transplant patients without andwith progression of coronary artery stenosis during a 3 years follow- upperiod. Heart transplant patients Without With progression progressionCharacteristics (n = 34) (n = 12) p-value Age of recipient (yr)  58 ±1.4  60 ± 1.4 **NS Gender of recipient (M/F) 21/14 11/1  *NS Age ofdonor (yr)  25 ± 1.3  32 ± 3.8 **NS Gender of donor (M/F) 27/7  10/2 *NS Duration of ischemia (min)  130 ± 6.7  140 ± 11  **NS No of HLAmismatches DR  1.2 ± 0.13  1.5 ± 0.15 **NS B + DR  2.8 ± 0.21  3.2 ±0.24 **NS Cytomegalovirus infection 24 11 *NS Hypertension 21 10 *NSDiabetes 1 2 *NS

[0180] TABLE 5 Laboratory data of heart transplant patients without andwith progression of coronary artery stenosis during a 3 years follow- upperiod. Heart transplantation patients Without With progressionprogression Characteristics (n = 34) (n = 12) p-value Serumtriglycerides (mg/dl)  130 ± 8.6  150 ± 14  NS HDL cholesterol (mg/dl) 50 ± 2.7  49 ± 4.9 NS LDL cholesterol (mg/dl)  110 ± 3.6   105 ± 8.7 NS Oxidized LDL (mg/dl)  1.2 ± 0.069  2.6 ± 0.33 0.0005

[0181] TABLE 6 Mild chronic Severe chrome renal failure Controls renalfailure non-dialysed hemodialysed Characteristics (n = 27) (n = 20) (n =21) (n = 56) Males/females 12/15 11/9 7/14 33/23 Age (years)  54(50-58)*  52 (44-60)*  55 (49-62)* 61 (58-65)* Body weight (kg)  72(69-76)*  73 (67-79)*  59 (53-65)* 65 (61-68)* Creatinine clearance 110(110-120)* 34 (29-39)* 8.4 (7-10)*  nd (ml/min) Primary renal disease:Glomerulonephritis — 4 3 11 Autosomal dominant polycystic — 2 6 10kidney disease Diabetes — 1 4 6 Reflux-nephropathy — 1 2 2 ChronicInterstitial Nephritis — 2 2 9 Hypertensive nephropathy — 2 0 2 Other¹ —6 1 9 Unknown — 2 3 7 Hypertension 1 16 19 18 Atherosclerotic heartdisease — 6 7 24 Cerebrovascular accidents — 0 3 9 Peripheral vasculardisease — 2 1 13

[0182] Laboratory data of study subjects Controls MCRF patients SCRFpatients HEMO patients (n = 27) (n = 20) p vs controls (n = 21) p vscontrols (n = 56) p vs controls Triglycerides (mg/dl)  120 (100-150) 150(130-170) NS 120 (100-140) NS 130 (110-160) NS HDL cholesterol (mg/dl)  44 (39-48)  38 (33-42) NS  44 (38-50) NS  37 (35-40) <0.05  LDLcholesterol (mg/dl)  120 (110-130) 110 (100-130) NS 110 (105-130) NS 120(110-130) NS Oxidized LDL (mg/dl) 0.59 (0.52-0.66)  1.6 (1.0-2.2) <0.01 1.8 (1.3-2.3) <0.001  3.2 (2.7-3.7) <0.001 Autoantibodies (titer)  4.2(4.0-4.4)  4.7 (4.0-5.4) NS  5.0 (4.2-5.8) NS  6.6 (5.7-7.4) <0.001 vWF(percent)  100 (90-110) 150 (110-180) NS 160 (130-190) <0.01 210(180-240) <0.001

[0183] TABLE 8 Characteristics of native LDL and of OxLDL isolated fromplasma of severe chronic renal failure patients Native LDL OxLDLProtein/antigen ratio >100 1.1 Reactivity with mAb-4E6 (C₅₀ mg/dl) 250.02 Relative electrophoretic mobility 1 1.7 Malondialdehyde (mole/moleprotein) 3 68 Substituted lysines per apo B-100 5 130Cholesterol/protein ratio 1.8 3.3 Free cholesterol/cholesterol esterratio 0.38 0.36 Phospholipid/protein ratio 1.7 1.6 Fatty acids (%) 16:014 37 18:1 19 50 18:2 55 10 20:4 12 3

[0184] TABLE 9 Multiple regression analysis of the dependence of OxLDLon the extent of renal failure Variable F-value p-value Age 1.2 0.28 Sex1.4 0.25 Hypertension 1.1 0.31 Triglycerides 1.5 0.23 HDL cholesterol1.7 0.20 LDL cholesterol 0.99 0.32 Renal failure 14 0.0004

[0185] TABLE 10 Multiple regression analysis of the dependence of OxLDLon hemodialysis and LDL cholesterol levels in severe chronic renalfailure patients Variable F-value p-value Age 0.31 0.58 Sex 0.19 0.66Hypertension 0.01 0.95 HDL cholesterol 0.02 0.89 Triglycerides 3.7 0.060Hemodialysis 5.6 0.021 LDL cholesterol 7.1 0.0095

[0186] TABLE 11 Inter-assay variation coefficients of sandwich ELISAusing 10 subsequent, independent MDA-modified LDL preparationsInter-assay variations Concentration coefficients (nM) (%) 10 9.6 5 7.62.5 8.4 1.25 13.2 0.62 12.0 0.31 13.0 0.16 12.3 0.08 15.5 0.04 16.9 0.0213.6 0.01 11.4

DOCUMENTS

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1. An immunological assay for the detection and/or quantification of MDA-modified LDL and OxLDL in a sample, said assay comprising: (a) contacting the sample with a first antibody that has high affinity for MDA-modified LDL and OxLDL; and (b) thereafter visualizing and/or quantifying a binding reaction between the first antibody and the MDA-modified LDL and OxLDL present in the sample; wherein the MDA-modified LDL and OxLDL for which the first antibody has high affinity contain at least 60 substituted lysine moieties per apo B-100 moiety.
 2. The assay of claim 1 in which the MDA-modified LDL and OxLDL for which the first antibody has high affinity contain at least about 90 substituted lysine moieties per apo B-100 moiety.
 3. The assay of claim 1 in which the MDA-modified LDL and OxLDL for which the first antibody has high affinity contain at least about 120 substituted lysine moieties per apo B-100 moiety.
 4. The assay of claim 1 in which the MDA-modified LDL and OxLDL for which the first antibody has high affinity contain at least about 210 substituted lysine moieties per apo B-100 moiety.
 5. The assay of claim 1 in which the MDA-modified LDL and OxLDL for which the first antibody has high affinity contain at least about 240 substituted lysine moieties per apo B-100 moiety.
 6. The assay of any of claims 1 to 5 which is a competitive assay.
 7. The competitive assay of claim 6 in which MDA-modified LDL and/or OxLDL are bound to a substrate, comprising contacting the sample and the first antibody with the substrate having bound to it MDA-modified LDL and OxLDL.
 8. The assay of any of claims 1 to 5 which is a sandwich assay in which the first antibody is bound to a substrate, comprising contacting the sample with the substrate having bound to it the first antibody.
 9. The assay of claim 8 in which a second antibody is used and the second antibody has high affinity for MDA-modified LDL and OxLDL.
 10. The assay of claim 9 in which the second antibody has high affinity for native LDL.
 11. The assay of any of claims 1 to 5 which is an immunohistochemical assay in which the sample is a tissue sample and it is contacted with the first antibody.
 12. The assay of any of the preceding claims in which the affinity constant of the first antibody for MDA-modified LDL and for OxLDL is at least about 1×10¹⁰ M⁻¹.
 13. The assay of any of the preceding claims in which the first antibody has low affinity for native LDL.
 14. The assay of claim 13 in which the affinity constant of the first antibody for native LDL is less than about 1×10⁶M⁻¹.
 15. The assay of any of the preceding claims in which the first antibody is the monoclonal antibody mAb-4E6 produced by hybridoma Hyb4E6 deposited at the BCCM under deposit accession number LMBP 1660 CB on or about Apr. 24,
 1997. 16. The assay of any of claims 8 to 10 in which the affinity constant of the second antibody for MDA-modified LDL and for OxLDL is at least about 1×10¹⁰M⁻¹.
 17. The assay of claim 16 in which the affinity constant of the second antibody for native LDL is at least about 1×10⁹M⁻¹.
 18. The assay of claim 17 in which the second antibody is the monoclonal antibody mAb-8A2 produced by hybridoma Hyb8A2 deposited at the BCCM under deposit accession number LMBP 1661 CB on or about Apr. 24,
 1997. 19. The assay of any of the preceding claims in which the sample is derived from the fluid or tissue of a human being.
 20. An immunological sandwich assay for the detection and/or quantification of MDA-modified LDL in a sample in which assay a first antibody that has a high affinity for MDA-modified LDL is bound to a substrate, said assay comprising: (a) contacting the sample with the substrate having bound to it the first antibody under binding conditions so that at least some of any MDA-modified LDL in the sample will bind to the first antibody; (b) thereafter removing unbound sample from the substrate; (c) thereafter contacting the substrate with a second antibody that has a high affinity for MDA-modified LDL; and (d) thereafter visualizing and/or quantifying the MDA-modified LDL that was present in the sample; wherein the MDA-modified LDL for which the first antibody and the second antibody have high affinity contains at least 60 substituted lysine moieties per apo B-100 moiety.
 21. The assay of claim 20 in which the MDA modified LDL for which the first antibody and the second antibody have high affinity contains at least about 90 substituted lysine moieties per apo B-100 moiety.
 22. The assay of claim 20 in which the MDA-modified LDL for which the first antibody and the second antibody have high affinity contains at least about 120 substituted lysine moieties per apo B-100 moiety.
 23. The assay of claim 20 in which the MDA-modified LDL for which the first antibody and the second antibody have high affinity contains at least about 210 substituted lysine moieties per apo B-100 moiety.
 24. The assay of claim 20 in which the MDA-modified LDL for which the first antibody and the second antibody have high affinity contains at least about 240 substituted lysine moieties per apo B-100 moiety.
 25. The assay of any of claims 20 to 24 in which the first antibody also has high affinity for OxLDL.
 26. The assay of any of claim 20 to 25 in which the first antibody has low affinity for native LDL.
 27. The assay of any of claims 20 to 24 and 26 in which the first antibody has low affinity for OxLDL.
 28. The assay of any of claims 20 to 27 in which the second antibody has high affinity for native LDL.
 29. The assay of any of claims 20 to 28 in which the affinity of the first antibody for MDA-modified LDL is at least about 1×10¹⁰M⁻¹.
 30. The assay of any of claims 20 to 29 in which the affinity of the first antibody for native LDL is less than about 1×10⁶M⁻¹.
 31. The assay of any of claims 20 to 30 in which the affinity of the second antibody for native LDL is at least about 1×10⁹M⁻¹.
 32. The assay of any of claims 20 to 26 and 28 to 31 in which the first antibody is the monoclonal antibody mAb-4E6 produced by hybridoma Hyb4E6 deposited at the BCCM under deposit accession number LMBP 1660 CB on or about Apr. 24,
 1997. 33. The assay of any of claims 20 to 24 and 26 to 31 in which the first antibody is the monoclonal antibody mAb-1H11 produced by hybridoma Hyb1H11 deposited at the BCCM under deposit accession number LMBP 1659 CB on or about Apr. 24,
 1997. 34. The assay of any of claims 20 to 33 in which the second antibody is the monoclonal antibody mAb-8A2 produced by hybridoma Hyb8A2 deposited at the BCCM under deposit accession number LMBP 1661 CB on or about Apr. 24,
 1997. 35. Monoclonal antibody mAb-4E6 produced by hybridoma Hyb4E6 deposited at the BCCM under deposit accession number LMBP 1660 CB on or about Apr. 24,
 1997. 36. Hybridoma Hyb4E6 deposited at the BCCM under deposit accession number LMBP 1660 CB on or about Apr. 24,
 1997. 37. Monoclonal antibody mAb-8A2 produced by hybridoma Hyb8A2 deposited at the BCCM under deposit accession number LMBP 1661 CB on or about Apr. 24,
 1997. 38. Hybridoma Hyb8A2 deposited at the BCCM under deposit accession number LMBP 1661 CB on or about Apr. 24,
 1997. 39. A stable standard containing MDA-modified LDL whose extent of substitution of its lysine moieties will remain essentially constant over normal periods of time during normal, storage for biological materials, the MDA-modified LDL of said standard being made by contacting malondialdehyde with LDL at a predetermined molar ratio of malondialdehyde to the apo B-100 moiety of the LDL, the standard containing an agent that reduces the ability of any metal ions present to catalyze oxidation of the LDL and/or an anti-oxidant.
 40. The standard of claim 39 wherein both an agent that reduces the ability of any metal ions present to catalyze oxidation of the LDL and an anti-oxidant are present.
 41. The standard of any of claims 39 to 40 wherein the agent that reduces the ability of any metal ions present to catalyze oxidation of the LDL is a chelating agent.
 42. The standard of claim 41 wherein the chelating agent is EDTA.
 43. The standard of any of claims 39 to 42 wherein the anti-oxidant is selected from the group consisting of BHT and Vitamin E.
 44. The standard of any of claims 39 to 43 further comprising a physiological fluid.
 45. The standard of claim 44 wherein the physiological fluid is plasma.
 46. The standard of any of claims 39 to 45 further comprising at least one anti-platelet compound and/or anti-coagulant.
 47. A stable calibrator for assays for MDA-modified LDL comprising the standard of any of claims 39 to
 46. 48. A stable control for assays for MDA-modified LDL comprising the standard of any of claims 39 to
 46. 49. A stable calibrator for assays for OxLDL comprising the standard of any of claims 39 to
 46. 50. A stable control for assays for OxLDL comprising the standard of any of claims 39 to
 46. 51. A kit for conducting a sandwich assay for the determination of OxLDL or MDA-modified LDL or both in a sample, said kit comprising: (a) a substrate on which is bound a first antibody that has high affinity for OxLDL or MDA-modified LDL or both, the OxLDL and MDA-modified LDL each having at least 60 substituted lysine moieties per apo B-100 moiety, and (b) a labeled antibody having a high affinity for OxLDL that becomes bound to the first antibody during the assay or for MDA-modified LDL that becomes bound to the first antibody during the assay or for both that become bound to the first antibody during the assay.
 52. The kit of claim 51 further comprising a reactive substance for reaction with the labeled antibody to give an indication of the presence of the labeled antibody.
 53. The kit of claim 51 wherein the reactive substance comprises an enzyme.
 54. The kit of any of claims 51 to 53 further comprising the stable calibrator of any of claims 47 or
 49. 55. The kit of any of claims 51 to 54 further comprising the stable control of any of claims 48 or
 50. 